EIKCS 2022: Biomarker Discovery Through Single-cell RNA Sequencing

(UroToday.com) In the tenth session of the 2022 International Kidney Cancer Symposium (IKCS): Europe meeting focusing on biomarker discovery in renal cell carcinoma (RCC), Dr. David Braun discussed the potential of single-cell RNA sequencing to facilitate biomarker discovery. In doing so, he emphasized how single-cell RNA sequencing can improve our understanding of the underlying biology of RCC, with important implications for both biomarker discovery and therapeutics.

To begin, he emphasized the importance of this single-cell based analysis. In contrast to bulk RNA sequencing in which the heterogeneity of tumor and related cell types and their states are lost, single-cell analyses allow a more focused approach that avoids the homogenization of cells which may dilute or obscure important findings. He noted that, in the context of RCC, this is a rapidly evolving field with much of the important work having been published in the last year or so.

In this context, he emphasized his own work addressing what he termed a “fundamental question” in biology of how the immune microenvironment evolves with advancing RCC stage. To do so, he and his team prospectively collected samples across spectrum of disease from localized to locally advanced and metastatic disease. Based on single-cell sequencing, they identify two major populations of T cells: the exhausted T cell population increases with increasing stage. Among myeloid cells, the authors observed changes in macrophages from pre-inflammatory to immunosuppressive-like markers, with enrichment of M2-like genes seen in macrophages of metastatic tumors. 

Building on these data, the authors inferred cell-cell interactions between these cell types. Importantly, they found that this was a bi-directional interaction. This allowed development of a signature (high CD8-TAM interaction signature) which was associated with a worse prognosis.

Summarizing data from Dr. Charles Drake, Dr. Braun emphasized that C1Q+ macrophages are enriched in kidney cancer, compared to the normal kidney. However, even further, they are associated with disease recurrence following initial therapy. Dr. Braun then moved to describe further work examining the effect of immune checkpoint inhibition on the tumor immune microenvironment. Based on single cell sequencing, there is evidence of upregulation of T cells and of macrophage checkpoints after immune checkpoint inhibition. Further, there is remodeling of T cell populations, particularly of the CD8+ T cell state, following immune checkpoint inhibition. These cells, when bound with anti-PD-1 antibodies, express granzyme, PD-1, and TIM-3. While it is helpful to understand changes that result following immune checkpoint inhibition therapy, it would be more clinically meaningful if we could predict response, instead of just looking at what happens in response to therapy. Using multi-regional sampling, Dr. Braun highlighted that there a divergent immune population in immune checkpoint inhibition-responsive and resistant tumors: resistant tumors are enriched in myeloid populations while responsive tumors show enrichment with tissue-resident memory like CD8+T cells.

Moving forward, he suggested that single cell RNAseq may be leverage for biomarker discovery by “building it into clinical trials”. In this model, he suggested that this approach would not be suitable for all patients enrolled in a trial but rather that a subset of patients would undergo a “deep” analysis using high-dimensional tools for a subset of patients on therapy then go back and try to validate using more conventional tools. This would allow for a more targeted discovery approach followed by a more generalizable validation. 

In terms of operationalizing this, he highlighted the opportunity of the ADAPTeR trial as previously discussed by Dr. Samra Turajlic, as well as ongoing collaborations he has to gain access to data from the HCRN GU16-260 and COSMIC-313 clinical trials.

He emphasized that to better inform improved diagnostic approaches and newer therapeutic paradigms, we need to better understand the disease. In the era of immune checkpoint inhibition, he suggested that we need to start with a patient’s tumor, dissect and micro-dissect it in the lab, learn about how treatment with immune checkpoint inhibition changes the tumor microenvironment, and then examine what tumor antigens are driving response. All of this must be considered in a spatial context that acknowledges the heterogeneity of RCC.

In conclusion, Dr. Braun states that single cell RNA sequencing allows the discovery of cell types, transcriptional states, and cellular interactions that are not apparent and may be “averaged out” using traditional bulk genomic analyses. Already, this approach has led to substantial advances in our understanding of the immunobiology of RCC. Moving forward, to enable biomarker discovery, he advocated that single cell RNA sequencing should be integrated into discovery cohort within clinical trials. Future work leveraging this single cell transcriptomic analysis in combination with better understandings of RCC antigen and spatial phenotypes may faciliate the transfer of discovery to improved biomarkers and therapeutics in clinic.